Throughout this application, various publications are referenced by Arabic numerals in brackets. Full citations for these publications may be found listed at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as of the date of the invention described and claimed herein.
There is an urgent need for better prognostic indicators to guide the vigor and extent of surgical and adjuvant therapies of patients, especially those with early stage, node negative breast cancer [1], and those with early stage prostatic cancer. One breast-cancer marker that is associated with an aggressive phenotype is ErbB2, a member of the EGF family of growth factor receptors [2]. High expression of ErbB2 in node positive patients generally predicts a poor short term clinical outcome [3-5].
Recent studies on breast cancers also implicate additional growth-factor receptors such as other members of the EGF-receptor family; hepatocyte growth factor (HGF) and its receptor, c-Met; IGF-1 and its receptor; FGFs and their receptors; mammary-derived growth factor (MDGF-1 and its receptor); and non-receptor tyrosine kinases c-Src and Brk [reviewed in 6, and see 7, 8-10]. In addition to serving as markers for aggressive breast cancer, these growth factor receptors may have important functional roles in the aggressive phenotype. To the extent that this is true, measures of the sum total of growth-factor receptor signaling in tumor cells might provide the urgently needed accurate prognostic ability to guide surgical, radiation and chemoendocrine adjuvant therapy.
ErbB2 is a 185,000 molecular weight transmembrane glycoprotein that has an extracellular ligand binding domain and a cytoplasmic domain with tyrosine kinase activity [11]. What distinguishes ErbB2 from other receptor tyrosine kinases is that it is active when over expressed, even in the absence of any ligand [12]. For this reason, cells that over express high levels of ErbB2 protein proliferate in the absence of serum, and frequently appear transformed [13]. Mechanistically, over expression allows stable receptor dimers to form [14]. In contrast, most other growth-factor receptors must first bind their ligand before they can productively dimerize [15]. Dimerization activates the receptor tyrosine kinase. ErbB2 can also form active heterodimers with other family members after they have bound ligand [12]. For example, the EGF receptor-family members, HER-3 and HER-4 bind to HRG/NDF (heregulin and neu differentiation factor) ligand and activate ErbB2 through transmodulation [16-18]. Similarly, ErbB2 forms heterodimers with EGF receptors bound to EGF, thereby activating the ErbB2 tyrosine kinase [12].
The activated ErbB2 tyrosine kinase phosphorylates itself on specific tyrosine residues. Several “second messenger” proteins via their SH2 and PTB domains recognize and bind to the receptor's phosphorylated tyrosines [19-21]. Many of these proteins are then tyrosine phosphorylated by the receptor, and thereby activated, propagating the signaling cascade. One of these second messengers is the adapter protein, She, which appears to help transmit the signal to Ras, and thereby ultimately to DNA synthesis and cell proliferation (see FIG. 1) [22, 23] (and also reportedly signals to Myc [24, 25] and to PT 3′ kinase [26]). The receptors phosphorylate tyrosine 317 in Shc, which in turn is recognized by Grb2-SOS complexes. As a result, SOS is translocated to the cellular membrane which appears to facilitate its ability to activate Ras [27-34]. It has been shown that signaling pathways to Ras are constitutively activated in many cell lines derived from breast cancers [35-38]. Further, several studies using microinjected antibodies to Shc, Shc antisense, and various Shc dominant-negative constructs have shown the dependence on a functional Shc of signaling from the EGF receptor, Her2/Neu, IGF-1 and HGF [8, 22, 39-42]. There are three isoforms of Shc: p66, p52 and p46 of 66, 52, and 46 kDa, respectively [21, 23, 43]. The p66 Shc isoform contains a unique N-terminal domain (CH2) not found in the p52 or p46 Shc isoforms [23]. In contrast to p52 and p46 Shc, p66 Shc does not activate the MAP kinase signaling cascade but rather actually inhibits the ability of growth factors to activate c-fos [44].
Currently the National Cancer Institute lists more than 95 open or planned clinical trials employing a myriad of tyrosine kinase inhibitors (TKI) specific for Her-2/neu, the EGF receptor or any of several other receptor and non-receptor tyrosine kineses. However, these trials in general are greatly hampered by the clinician's inability to predict which patients have tumors that are likely to respond to any single TKI or combination of TKIs.